Membrane proteins are critical components of many fundamental biological processes, enabling cell signaling and material and energy transduction across cellular boundaries.1 As such, their malfunction has been linked to numerous diseases and they are common targets for pharmacological treatments.2 However, rational drug design has been limited by difficulties in obtaining high resolution structural information on these proteins.
The key bottleneck in the determination of membrane protein structures is the identification of appropriate crystallization conditions. These proteins are typically available in quantities that are insufficient to screen a large number of conditions.3 Additionally, they exhibit poor solubility due to their amphiphilic nature.1,4 As a result, a tremendous disparity has developed between the number of known structures for membrane proteins (˜368) as compared to soluble, globular proteins (>50,000).5,6 
In recent years, microfluidic technology has been successfully utilized for high throughput screening of crystallization conditions at the nanoliter scale or smaller.3,7 Thus far crystallization of membrane proteins in microfluidic systems has been limited to in-surfo methods where detergents are used to solubilize membrane proteins and crystallization is attempted as for soluble proteins.3,8 
While traditional microfluidic devices have often experienced difficulties in dealing with highly viscous, complex, or congealing fluids, a method of two-phase flow has been able to handle this. In this method, droplets are isolated from the surrounding walls by means of a carrier fluid and are mixed internally by viscous forces. In this manner it is able to deal with viscous or congealing materials such as blood.3,53 The droplet mixer, while able to deal with more viscous fluids, still requires the flow of all materials for the formation of droplets. It is also limited by fluid property requirements for the formation of these droplets. Furthermore, while droplets containing water and lipid can be formed, the shear forces present in droplet-based mixing are inadequate to drive mixing of these materials to form mesophases.
An alternative, in-meso crystallization method (also referred to as cubic lipidic phase cyrstallizatin or in-cubo crystallization) uses an artificial aqueous/lipid mesophase to maintain membrane proteins in a membrane-like environment.1,4 This method exploits the complex phase behavior of aqueous/lipid systems (e.g. lamellar, bicontinuous cubic phases),9,10 creating local variations in the curvature of the bilayers to drive crystal nucleation and growth.1,4,10-13 Despite its benefits, implementation of the in-meso approach to crystallization on the microscale has been particularly difficult. To this point aqueous/lipid mesophases necessary for the in-meso approach have been prepared either by centrifugation,12 or using coupled microsyringes; FIG. 1 illustrates coupled microsyringes having a volume of ≧20 μL.14 Unfortunately both methods require quantities of purified membrane protein (10-500 μL) that are potentially inaccessible or undesirable.
Creation of the necessary lipidic mesophases at much smaller scales, for example using microfluidics, is particularly challenging due to the ˜30-fold difference in the viscosities of the pure components: 2.45×10−2 versus 7.98×10−4 Pa-s for the monoolein lipid phase (1-monooleoyl-rac-glycerol) and the aqueous phase, respectively or the ˜60,000-fold difference in the viscosity of the aqueous phase and the resulting mesophase (˜48.3 Pa-s at a shear rate of 71.4 s−1). Moreover, the resulting mixture exhibits highly non-Newtonian behavior.15,16 The highly viscous and non-Newtonian nature of the fluids render previously reported mixing approaches ineffective.17,18 